Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus.

To study adaptive evolution in defined environments, we performed evolution experiments with Saccharomyces cerevisiae (yeast) in nitrogen-limited chemostat cultures. We used DNA microarrays to identify copy-number variation associated with adaptation and observed frequent amplifications and deletions at the GAP1 locus. GAP1 encodes the general amino acid permease, which transports amino acids across the plasma membrane. We identified a self-propagating extrachromosomal circular DNA molecule that results from intrachromosomal recombination between long terminal repeats (LTRs) flanking GAP1. Extrachromosomal DNA circles (GAP1(circle)) contain GAP1, the replication origin ARS1116, and a single hybrid LTR derived from recombination between the two flanking LTRs. Formation of the GAP1(circle) is associated with deletion of chromosomal GAP1 (gap1Δ) and production of a single hybrid LTR at the GAP1 chromosomal locus. The GAP1(circle) is selected following prolonged culturing in L-glutamine-limited chemostats in a manner analogous to the selection of oncogenes present on double minutes in human cancers. Clones carrying only the gap1Δ allele were selected under various non-amino acid nitrogen limitations including ammonium, urea, and allantoin limitation. Previous studies have shown that the rate of intrachromosomal recombination between tandem repeats is stimulated by transcription of the intervening sequence. The high level of GAP1 expression in nitrogen-limited chemostats suggests that the frequency of GAP1(circle) and gap1Δ generation may be increased under nitrogen-limiting conditions. We propose that this genomic architecture facilitates evolvability of S. cerevisiae populations exposed to variation in levels and sources of environmental nitrogen.

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator.

Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Deltasnf1, Deltasnf4, and Deltasnf1Deltasnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases.

Integrative analysis using proteome and transcriptome data from yeast to unravel regulatory patterns at post-transcriptional level.

Exist several studies on the correlation between proteome and transcriptome and these studies have shown that generally there is only a weak positive correlation between these two omes, which means that post-transcriptional events play an important role in determining the protein levels in the cell. In this study we combined proteome and transcriptome data from six different published dataset to identify patterns that can provide new insight into the reasons for these deviations. By using a categorization method and integrating genome-scale information we found that the relation between protein and mRNA is related to the gene function. We could further identify that for genes belonging to amino acid biosynthetic pathways there is no translational regulation, meaning that there is generally a good correlation between mRNA and protein levels. We also found that there is generally translational control for large proteins and there also evidence for a role of conserved motifs in the 3' untranslated regions in the mRNA-protein correlation, probably by controlling the level of mRNA.

Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: a three factor design.

BACKGROUND: Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae. The three factors we considered were specific growth rate, nutrient limitation, and oxygen availability. RESULTS: We identified 268 growth rate dependent genes, independent of nutrient limitation and oxygen availability. The transcriptional response was used to identify key areas in metabolism around which mRNA expression changes are significantly associated. Among key metabolic pathways, this analysis revealed de novo synthesis of pyrimidine ribonucleotides and ATP producing and consuming reactions at fast cellular growth. By scoring the significance of overlap between growth rate dependent genes and known transcription factor target sets, transcription factors that coordinate balanced growth were also identified. Our analysis shows that Fhl1, Rap1, and Sfp1, regulating protein biosynthesis, have significantly enriched target sets for genes up-regulated with increasing growth rate. Cell cycle regulators, such as Ace2 and Swi6, and stress response regulators, such as Yap1, were also shown to have significantly enriched target sets. CONCLUSION: Our work, which is the first genome-wide gene expression study to investigate specific growth rate and consider the impact of oxygen availability, provides a more conservative estimate of growth rate dependent genes than previously reported. We also provide a global view of how a small set of transcription factors, 13 in total, contribute to control of cellular growth rate. We anticipate that multi-factorial designs will play an increasing role in elucidating cellular regulation.

Physiological characterization of glucose repression in the strains with SNF1 and SNF4 genes deleted.

We investigated the effect of Snf1 kinase and its regulatory subunit Snf4 on the regulation of glucose and galactose metabolism in the yeast Saccharomyces cerevisiae by physiologically characterizing Deltasnf1, Deltasnf4 and Deltasnf1Deltasnf4 in CEN.PK background in glucose and glucose-galactose-mixture batch cultivations. The main result of this study showed that delayed induction of galactose catabolism was SNF1 or SNF4 gene deletion specific. In comparison to the reference strain, growth delay on galactose was found to last 2.4 times (7 h), 3.1 times (10.5 h) and 9.6 times (43 h) longer for the Deltasnf4, Deltasnf1 and Deltasnf1Deltasnf4 strains, respectively. The maximum specific growth rates on galactose were determined to be two to three times lower for the recombinant strains compared to the reference strain (0.13 h(-1)) and were found to be 0.07, 0.08 and 0.04 h(-1) for the Deltasnf1, Deltasnf4 and Deltasnf1Deltasnf4 strains, respectively. The study showed that Snf1 kinase was not solely responsible for the derepression of galactose metabolism.

Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains: the comparison of two quantitative methods.

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches was used to identify relative changes in the protein expression levels between the strains. A total of 2388 proteins were relatively quantified, and more than 350 proteins were found to have significantly different expression levels between the two strains of comparison when using the stable isotope labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. The functional categorization of the relative protein expression differences that occur in Snf1-deficient strains uncovers a wide range of biological processes regulated by this important cellular kinase.

Global transcriptional and physiological responses of Saccharomyces cerevisiae to ammonium, L-alanine, or L-glutamine limitation.

The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with l-glutamine, l-alanine, or l-ammonium in limitation and by growing cells in an excess of ammonium. Cells grown in l-alanine-limited cultures had higher biomass yield per nitrogen mole (19%) than those from ammonium-limited cultures. Whole-genome transcript profiles were analyzed with a genome-scale metabolic model that suggested increased anabolic activity in l-alanine-limited cells. The changes in these cells were found to be focused around pyruvate, acetyl coenzyme A, glyoxylate, and alpha-ketoglutarate via increased levels of ALT1, DAL7, PYC1, GDH2, and ADH5 and decreased levels of GDH3, CIT2, and ACS1 transcripts. The transcript profiles were then clustered. Approximately 1,400 transcripts showed altered levels when amino acid-grown cells were compared to those from ammonium. Another 400 genes had low transcript levels when ammonium was in excess. Overrepresentation of the GATAAG element in their promoters suggests that nitrogen catabolite repression (NCR) may be responsible for this regulation. Ninety-one genes had transcript levels on both l-glutamine and ammonium that were decreased compared to those on l-alanine, independent of the concentration. The GATAAG element in these genes suggests two groups of NCR-responsive genes, those that respond to high levels of nitrogen and those that respond to levels below 30 muM. In conclusion, our results reveal that the nitrogen source has substantial influence on the transcriptome of yeasts and that transcriptional changes may be correlated to physiology via a metabolic model.

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.